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Assay Method Information

Assay Name:  Radiometric Assay
Description:  Rat FAAH was prepared from male Sprague Dawley rat brains, homogenized in a potter in 20 mM of Tris HCl pH 7.4, 0.32 M sucrose.The radiometric assay used to measure FAAH activity was performed in Eppendorf tubes: 50 μg of total rat brain homogenate were pre-incubated in 445.5 μL of assay buffer (50 mM Tris-HCl pH 7.4, 0.05% Fatty acid-free-bovine serum albumin (BSA)) with 4.5 μL of inhibitor (at appropriate concentration in DMSO) or DMSO alone (to measure FAAH total activity) for 10 min at 37° C. The blank (no activity control) was prepared using 445.5 μL of assay buffer and 4.5 μL of DMSO without the 50 μg of total rat brain homogenate.After 10 min of pre-incubation with test compounds, the reaction was started by adding of 50 μL of substrate and incubating for 30 min at 37° C. The substrate was prepared in assay buffer in order to achieve the final concentration of 1 μM arachidonoyl ethanolamide (Cayman Chemical N. 90050) and 0.6 nM anandamide [ethanolamine-1-3H] (American Radiolabeled Chemicals Inc, ART. 0626, Conc. 1 mCi/mL, S.A. 60 Ci/mmol). The reaction was stopped by adding cold 1:1 CHCl3/MeOH. After 10 min of centrifugation (845×g at 4° C.) 600 μL of aqueous phase were transferred into scintillation vials previously filled with 3 mL of scintillation fluid (Ultima Gold™, Perkin Elmer Inc., Cat. 6013329). Radioactivity was measured by liquid scintillation counting (MicroBeta2 LumiJET Perkin Elmer Inc.).
Affinity data for this assay
 

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Last update November 1, 2007
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